AU618536B2 - Novel 3',4'-dinitrogen substituted epipodophyllotoxin glucoside derivatives - Google Patents

Description

I

\~53

AUSTRALIA

SPatents Act COMPLETE SPECIFICATION~

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Rela'L-d Art:.

This docurnent contains thle nnjjenjdmr. llwe under Section t3 by 1 6e Super" v~si;Ig Lxuniincr c .l and is 09 0 0049 *44 I t *9 I 4 I 9< 9 *1 I I I 9

I.

4 1

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IL

4 9 APPL.ICANT'S REFERENCE- CIT-1881.

Name(s) of Applicant(s): -Br-istol-Myers-Company 9t~ 6 t b 16 ,o ro rq n.

'76 A~ddress(es) of Applicant(s): 345 Park Avenue, New York, New York, UNITED STATES OF AMERICA.L Address for Service is: PHILLIPS OF14CNDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street MelW- urne 3000 AuIsTRALiA Complete Specification for the invention zntitled NMME 31,41 -DINITROGEN STJBSTrLUTED EPIPOOPHYLIDMflXN GIXCOSXtDVV

DERIVATIVES

our Ref 94520 POF Code: 1490/1490 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): K~F 6003q/2, .1

J

I

a I I I BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to novel dinitrogen substituted derivatives of epipodophyllotoxin glucosides, to their therapeutic anti-tumor use, and to pharmaceutical dosage forms containing these new agents.

2. scrip .on of the Related Art Etoposide (VP-16, la) and teniposide (VM-26, Ib) are clinically useful anticancer agents derived from the natuo rally occurring lignan, podophyllotoxin The numbering system used for nomenclature purpose is shown in Formula TI.

Etoposide and teniposide are epipodophyllotoxin

D

II

1", r f b: Y;ii C, V 4eQs Uco 0 0A

NCO

Y Cco 0 2 re k i cc 4 a 8 la: A=CH Ib: A=2-thienyl -2- I

I

derivatives; epipodophyllotoxin being the epimer of podophyllotoxin at the 4-position. Etoposide and tenip5.side are active in the treatment of a variety of cancers including small cell lung cancer, non-lyinphocytic leukemia, and non-seminomatous testiculaxc cancer (AMA Drug Evaluatlion, Edition, American Medical Asscvciation, 1983, Chicago, Illinois, p. 1554-5).

Etoposide and teniposide, and methods for producirig them, are discl~osed in US Patent 3,524,844 to Keller-Juslen et al. Etoposide 3',4'-guinone (IIla) has been generated from electrochemical oxidation of etoposide (Iolthuis J. J.

et al, J. Electroanal. Chem. Interfacial Electrochem., 1985, 184(2):317-29). The piaparation of the quinone III by chemical oxidation is disclosed in US patent 4,609,644 to Josef Nemec. Epipodophyllotoxin 3',4'-quinone derivatives III wherein A and Y have the definition given hereinbelow for Formula IV, serve as the starting material for our preparation of the nitrogen containing epipodophyllotoxin, derivatives of the present invention.

O,~

00 0 0000 0000 0 00*0 0000 0 0000 o *0 00 0 0 00 0 0 000 0 0 40 0 0 00 o *0 00 0 0 00 0 00 00 0 0 00 00 0 0 00 00 0 00 O 00 S 'L IlIa: A=CH 3

Y=H.

A

§Ayres and Lim in Cancer Chemother Pharmacol, 1982, 7:99-101 discloses the podophyilotoxin having the formula H 4 4 SUMMARY OF THE INVENTION The present invention, relates to antitum~or compounds h-aiing the formula IV

Y

IV

where in Y is H and A is selected from the group consisting of (C 1 10 )alkyl; (C 2 2 2 0 )alkenyl; (C 5 )cycloalkyl;.

2-furyl; 2-thienyl; aryl, aralkyl, and aralkenyl, -4i pr~- I wherein each of the aromatic rings may be unsubstituted or substituted with one or mcre groups selected from halo, (C 1 8 )alkyl, (C 1 8 alkoxy, hydroxy, nitro, and amino; or A and Y are each (C 1 8 )alkyl; or A and Y and the carbon to which they are attached join to form a cycloalkyl group; and B is selected from the group consisting of 4,40.

4, b44,.

I S I NOR z

J

31 -C K Hco^-r^~P M^Q-jK 5 rg 0.

3r

CI

c' 0 ,3IQ RL 1 3 i /cx gir~x 1 2 wherein R and R are independently selected from the group to consisting of (C1- 5 )alkyl, aryl, and aryl(C1- 5 alkyl; R 3 and

R

4 are independently H, (C 1 5 )alkanoyl, or halo-substituted (C2- 5 )alkanoyl; R is aryl, aryl substituted with one or

.I

-r more groups selected from (Cl ))alkoxy and nitro, or 6 7 heteroaryl; R and R are each H or (C 1 5 )alkyl;

R

8 is (C 1 5 )alkyl or (C 1 5 )alkyl substituted with one or more groups selected from the group consisting of hydroxy, alkoxy, alkanoyloxy, cyano, amino, alkylamino, dialkylamino, carboxy, alkylthio, mercapto, alkanoylamino, alkanoyl, carbamoyl, and halo; and X is oxygen or sulfur. It is to be understood that the structural formulas representing the B substituent depicted in the specification and in the claims are meant to encompass all diastereomeric and/or tautomeric forms where such are possible.

The present invention also relates to a process for preparing a compound of formula IV which comprises the steps of: reacting a compound of formula (1) with at least 1 molar equivalent each of a compound of the formula H2NOR 1 and dan 2NOR 2 or anacid addition salt thereof, to yield Sa, compound of formula (2) 77o\ 6 u e i)

Y

HO OH 00 1O (2) a o 0 CH 0 NOR 2

NOR

1 wherein A, Y, R and R are as previously defined and R and R may be the same or different; optionally hydrogenating a compound of formula Q in the presence of a noble metal catalyst to yield 1a compound of formula (3) 0 and optionally reacting a compound of formula (3) "with at least one molar equivalent each of a compound of formula L-R 3 and L-R 4 wherein L

S

3 is a leaving group, R 3 and R are the same or different selected from (C_1 5 )alkyl; in the presence of an acid acceptor to provide a compound of formula (4) U 77 6A 3 4 is a'NQ l n uaae or di-rn-eece rm(- ~ly;i

CHO

3 ~A AL 9 0* OR I3

NIRI

or (ii) with at least 2 molar equivalents of a compound of the formula R 5C(O)H is as defined in Claim 1, to yield a compound of formula

OHH

N=HR

or (iii) with a nitite i the presence of an acid to provide a compound of formula (6) 9.

000 co 0 39 6B -6cor (iv) with a compound of formula R C(O-alk) 3 wherein R is as defiid in Claim 1, and in the presence of an acid to provide a compound of formula (7' 0 I^

R

or with a compound of the formula R6C(O)C(O)R wherein R 6 and R are as defined in Claim 1, to provide a compound of formula Y or (vi) with a compound of formula L 2

-PO

2

R

8 wherein L 8 is a leaving group and R is as defined in Claim 1, and in the presence of an acid acceptor to provide a compound of formula (9) 0\ 0 CH 0 1 DETAILED DESCRIPTION OF THE INVENTION SThe starting material for the present invention, the 3',4'-quinone III may be prepared by reacting an oxidizing agent with a r'-demethylepipodophyllotoxin- -D-glucoside derivative I. The method is described in US Patent 4,609,644 which is hereby incorporated by reference.

One aspect of the present invention provides bis-oxime ethers of Formula V wherein, A,Y, R and R are as defined above

Y

0 0 C(v)

NO

1 s0 A preferred embodiment provides compound of 1 2\ S Formula V wherein R and R are selected from the grOup consisting of (C 5 )alkyl and aryl(Cl_ 5 alkyl, with methyl and phenylmethyl being the most preferred groups.

6D *77 r-6 Bis-oxime ethers of Formula V may spared by reacting an 3',4'-quinone III with an excess amount of an O-substituted hydroxylamine, or an acid addition salt thereof, in a suitable organic solvent such as pyridine.

The reaction is preferably carried out at elevated temperature for a period sufficient to convert the starting material to the bis-oxime ether; typically such reaction time is 24 hours or more. The products thus formed may be isolated and purified by conventional techniques e.g. flash o chromatography; or alternatively, they may be reduced directly, without first being isolated, to the corresponding 3',4'-diamino compound of Formula VI.

Accordingly, a further aspect of the present invention provides the diamino compound of Formula VI wherein A and Y are as previously defined, and pharmaceutically acceptable o\ .acid addition salts thereof.

4 0 4 Y 4*Y A- 1, o 4 40 0 44 4 4" "4

VI

The diamine of Formula VI may be prepared by reduction of the bis-oxime ether of Formula V; and as mentioned abcve, either a purified compound of Formula V or the crude product 1)O may be used. Reduction of the bis-oxime ether may be effected by conventional methodologies, e.g. a mild chemical -7- -i~i~i n-I i i reducing agent, or hydrogenation in the presence of a suitable catalyst such as Pt, Pd, Ni, R] or Rh. Catalytic hydrogenation is preferably employed.

SThe diamino compounds Formula VI may be further Sderivatized to provide for example, amides, imines, and heterocyclic compounds as defined for Formula IV. The reactions are generally carried out in inert organic solvents such as tetrahydrofuran, dichloromethane, or chloroform, under conditions that are appropriate for (I achieving the desired products. Products may be isolated and purified using known methods such as recrystallization and various chromatographic techniques.

Thus, according to another aspect of the invention 3 4 amides of Formula VII are provided wherein A, Y, R and R are as previously defined, except R 3 and R are not both H.

V

C34

VVII

VII wherein R 3 and R 4 are both (C 5)alkatoyl or halo-substituted (C_)alkanoyl.

-8- H f, Amide derivatives may be prepared by conventional acylating methodologies well known to a person of ordinary skill in synthetic organic chemistry. Suitable acylating agents include, but are not limited to, carboxylic acid, preferably in the presence of a condensing agent such as dicyclohexylcarbodiimide (DCC); an acid halide; a symmetrical or unsymmetrical anhydride; or a reactive ester or amide. In general, in preparing amide derivatives using an acid halide or an anhydride the reactions are preferably S carried out at below room temperature and in the range of from about -20 0 C to about 10 0 C. In the foregoing discussion, bis acylated derivatives are preferentially obtained when the reactic-i is carried out in the presence of I a base and when the acylating agent is used in a molar re* amiount at least twice that of the diamine compound of Formula VII; suitable bases are e.g. pyridin-, triethylamine, diisopropyl ethylamine, and dimethylaminopyridine. When the acylating agent is used in an amount equivalent to that of the diamine compound and sLo without the base, a mixture of and 4'-mono acylated derivatives are obtained.

According to another aspect of the present invention, there are provided bis-iminio compounds of Formula VIII wherein A, Y, and R are as previously defined.

-9- LU i i

,Y

OH

K K-

'VIII

Bis-imino compounds of Formula VIII may be formed when diamino compounds of Formula VI are reacted with an excess amount of aldehyde at room temperature preferably in.the presence of an acid catalyst such as p-toluenesulfonic acid, and also preferably employing a method for water removal; suitable methods therefor include the use of a dehydrating o agent such as molecular sieves, or the use of azeotropic -distillation. Compounds of Formula VIII are frequently labile, and a preferred method for their isolation is by 0* \Q chromatography using neutral alumina.

According to another aspect of the invention, there are provided compounds of Formula IV wherein B represents the heterocyclic groups IVa to IVd.

Thus, diazotization of 4',5'-diamino compounds of Formula VI provides the corresponding triazole derivative (Formula IVa). Imidazole derivatives (Formula IVb) may be -i d-3 A. POF Code: 1490/1490 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 6003q/ 1 prepared by reacting the diamino compounds with trialkyl orthoesters. Reaction of the diamino compounds with a 1,2-dioxo alkane provides pyrazine derivatives (Formula IVc). Re-ction of the diamino compounds with dihaloalkylphosphate or dihaloalkylthiophosphate in the presence of an organic base such as pyridine provides the corresponding cyclic phosphamide or thiophosphamide derivatives (Formula IVd) respectively.

BIOLOGICAL ACTIVITY Representative compounds of the present invention were evaluated for their antitumor activity in in vitro cytotoxicity assay against human and murine tumor cell lines, as well as against transplantable murine P388 leukemia.

4 o* P388 Leukemia.

o Female CDF 1 mice were implanted intraperitoneally with a tumor inoculum of 106 ascites cells of P388 murine leukemia and treated with various doses of a test compound; 4 four mice were used for each dose level axid ten were used as saline-treated control. The compounds were administered by *intraperitoneal injection on days 5 and 8 (day 1 being the day of tumor implantation). Antitumor activity was expressed as T/C which is the ratio of the median survival time (MST) of drug-treated group to the MST of saline-treated control group. A compound showing a T/C value of 125 or greater is generally considered to have significant antitumor activity in the P388 test. The experiment lasted 31 days at the end of which time thenumber of survivors was noted. Table I presents the results o0 of the above-described evaluation; only the maximum T/C and the dose showing the maximum effect are reported.

1_1 Table I. Antitumor activity against P388 Leukemia Compound of Example Dose (mg/kg/inj) 100 >200 >100 >160 >180 >180 >140 >180 >120 Max. T/C 126 175 (270) 216 105 (270) 145 (270) 125 (>370) 115 (270) 110 (270) 145 (270) 0 99 9, 0 c9Q4 0999 0~* 0 00 909000 0 0) The values in parentheses are the values obtained with etoposide as the positive control in the same experiment.

Cytotoxicity Assay The in vitro cytotoxicity assay involved growing o" various mammalian tumor cells, including human tumor cells, *on microtitra plates employing established tissue culture methods. The concentration of each compound required to 40 *o inhibit cell growth by 50% (IC 5 0 was then determined by a four-fold serial dilution technique. The validity of the method has been supported by a report published in the "Proceedings of the American Association for Cancer SResearch", 1984, 25:1891 (Abst. No. 328). Tumor cells of the following types were employed for each compound tested: B16-F10 murine melanoma; KB human rnsopharyngyl; Moser human colon; SW900 human lung; M109 murine lung; and three human colon tumor cell lines namely HCT-116, HCT-VM, and HCT-VP, the latter two being resistant to teniposide (VM) and 2o etoposide respectively. IC 5 0 values less than. 500 Pg/ml are a positive indicator of antitumor activity. Table -12- II presents IC 50 values of various compounds of the present invention against the aforementioned cell lines.

Table II In vitro cytotoxicity assay _I.Cr .values (lig/ml)* B-16-FlO HCT-116 HCT/VM46 HCT/VP35 MOSER SW900 Examrple 17 53 64 84 91 78 62 88 82 88 89 3-16-FlO HCT-116 MOSER SW900 Example 2 29 84 54 >250 44451 91 40 >250 14' Exa.nple 4 J.~8 116 >25 0 106 >250 98 >250 123 >250 Example 4 459 81 31 25 0 100 103 65 >250 Example 18 82 >250 116 >250 >250 115 >250 Example 4118 103 113 113 99 90 >250 Example 21 23 20 2.1 >250 17.9 26 23 >250 B-16-F1O HCT-116 KB MOSER M109 Example 1 14.5 7.4 37 43 83 -13- 2-furyl; 2-thienyl; aryl, aralkyl, and aralkenyl, -4p~ 1 j Table II cont'd 14.1 17.4 28 11 59 Example 3 59 108 2'90 87 104 146 102 91 91 87 From the data presented above it is observed that although some of the analogs tested do not show activity against in vivo P318 leukemia, they may all be considered active against various solid tumors in in vitro cytotoxicity 4 assays.

Accordingly, this invention provides a method for inhibiting tumor cell growth which comprises administering an effective tumor-inhibiting dose of an antitumor compound of formula IV to a tumor bearing host.

oAnother aspect of this invention provides a pharmaceutical composition which comprises an effective tumor-inhibiting amount of an antitumor compound of formula IV and a pharmaceutically acceptable carrier. These compositions may be made up of any pharmaceutical form S appropriate for the desired route of administration.

Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for or 1l administration such as solutions, suspensions, syrups or Selixirs and preprdtions for parenteral administration such as sterile solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, S physiological saline or some other sterile injectable medium o0 immediately before use.

Optimal dosages and regimens for a given mammalian host can be readily ascertained by those skilled in the art. It -14will, of course, be appreciated that the actual dose used will vary according to the particular composition formulated, the particular compound used, the mode of application and the particular site, host and disease being treated. Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease.

\o The following examples are for illustrative purposes only should not be construed'as limiting the scope of the invention.

In the following examples, all temperatures are given in degrees Centigrade. Melting points were recorded on a Thomas-Hoover capillary melting point apparatus and are 9 44 4 4 1 1 t's* uncorrected. H NMR spectra were recorded either on a Bruker WM 360 or a Varian VX2 200 spectrophotometer (using CDC1 3 as an internal reference). Chemical shifts are reported in 6 units and coupling constants in Hertz.

a, Splitting patterns are designated as follows: s, singlet; d, 4 44 doublet; t, triplet; q, quartet; m, multiplet; bp, broad peak; and dd, doublet of doublet. Infrared spectra were Sdetermined either on a Beckman Model 424C or"a Perkin-Elmer 1800 Fourier Transform Infrared Spectrophotometer and are -1 reported in reciprocal centimeters (cm Thin-layer chromatography (TLC) was carried out on precoated silica gel plates (60F-254) using UV light and/or iodine vapors as visualising agents. High and low resolution mass spectra were recorded on KRATOS MS 50 and KRATOS MS 3 Spectrophotometer,' respectively. "Flash Chromatography" refers to the method described by Still (Still, W.C. et al, J. Org. Chem., 1978, 43:2923) and was carried out using either E. Merck silica gel (200-400 mesh) or Woelm silicd, gel (32-63 pim). All evaporations of solvents were performed L I represent the structural fragment r O Example 1 4'-Dehydroxy-3'-demethoxy-etoposide 3',4'-bis-O-benzyloxime S*

ETOP"

SaI It i s Solid O-benzyl-hydroxylamine hydrochloride (2.80 g, 17.5 mmol) was added to a solution of etoposide 3',4'-quinone ,2.50 g, 4.37 mmol) in pyridine (75 ml). The mixture was stirred at 54 0 C for 43 hrs and then at 60-70 0 C for an additional 15 min. Pyridine was removed under reduced Ce pressure and the residue was dissolved in CH 2 C1 2 (250 ml) and extracted with a mixture of H20 (200 ml) and 1 N HC1 ml). The aqueous layer was further extracted with CH 2 C1 2 (2 x 15 ml) and the combined extracts were washed with H20 (100 ml) and brine ml) and dried over Na2SO 4 After evaporation of the solvent, 3.7 g of a yellow orange solid was obtained which was purified by flash chromatography on silica gel. Elution with 1% CH 3 OH in CH 2 C1 2 produced 2.10 g (61.41) of the pure title compound as a yellow orange solid, mp 152-156 0

C.

-16- 1 H NMR (CDCl 3 6 38-7. 11 10H) 6. 76 1H) 53 (s,lH) 6. 02 (d,1H) 5.96 (d,2H) 5.90 (d,1H) 5.46 (s,2H) 13 (s,211) 4. 82 (d,1H,J=3.5Hz) 4. 73 (q,1E,J=5Hz) 4. 61 (d,1H,J=7. 6Hz) 4. 42 (dd, 1H) 4. 30-4.24 (m,2H) 4. 14 (dd,1H,J=3. 9 and 10.4Hz) 3. 79 3H) 3. 74-3. 69 3. 57-3. 51 (m,l1H) 3. 43-3. 37 1H) 3. 32-3. 29 2H) 31.23 (d6d,1H,J=5. 5 and 14.1lHz) 2. 89-2. 82 1H) 2. 64 1H,J=2Hz ,OH) 2.30 1H,J=2. 3Hz, OH) 1. 37 3H,J=5Hz) Ex ampl1e 2 to' 4'-Dehydroxy-3 '-demethoxy-etoposide 3 4 '-bis--O-methyloxime *141 4(11 4 I 11 1 I Il 4(4(44 I 8 4 ~4 I I t I 48 I (4 41 1 4 4.

I *I 41 4 4411

"ETOP"

W-C3 Solid methoxylamine hydrochloride 0 q, 35.mmol) was added to a solution cf etoposide o-guinone (5.0 g, 8.73 mmol) in pyridine (50 ml) stirring at room temperature under N 2~ The dark red solution immediately changed to a dark orange solution upon addition. The reaction mixture was heated for 24 hours at 60 0 C and then for an additional 24 hours at 75 0 C. The pyridine was removed by evaporation in vacuo.

Flash chromatography on silica gel using 4% MeOH in CH 2 Cl 2 as eluent provided 4.73 g of yellow-orange solid mp 2O5-21,Dc I(KBr) 3480 2920, 1780, 1495 cm 1 HNMR (CDC1 3 6 6. 79 6. 53 1H) 6. 00 3H) 5. 83 3'i) 4. 88 J=3. 2Hz, 1H) 4. 75 4. 64 3. 79 3H) 3. 75-3. 20 6H) 2. 94 (m,1IH) 1. 38 OHz, 3H).

-17sl~l:

I

MS .(FAB) m/e 6'1 (M+II)+ Example 3 4'-Dehydroxy-3'-demethoxy-3',4'-diamino etoposide

"ETOP"

ao I A solution of 4'-dehydroxy-3'-demethoxy-etoposide 3',4'-bis-O-benzyloxime (2.00 g, 2.55 mmol) in reagent S. alcohol (100 ml) and ethyl acetate (65 ml) was treated with f t 20% palladium hydroxide on carbon and hydrogenated at 65 psi for 1.5 hrs. The mixture was filtered through a pad of Celite and washed with ethyl acetate. The filtrate was to concentrated and purified by flash chromatography on silica gel. Elution with 2% CH3OH in CH 2 C1 2 followed by 5% CH 3

OH

oin CH2C12 gave 1.12 g of the pure title compound as an off-white solid, mp 235-240 0 C (dec, darkens at 200 0

C).

Trituration with ether gave the analytical sample.

IR (KBr) 3420, 1775, 1505, 1485, 1230, 1165, 1095, 1075, a 1040, 1005, 930 cm SH NMi 'CDC13) 6 6.78 6.54 6.35 (d,1H,J=1.3Hz), 5.95 5.70 (d,1H,J=1.3Hz), 4.86 (d,lH,J=3.3Hz), 4.73 (q,1H,J=5Hz), 4.63 (d,1H,J=7.6Hz) 4.52 a (d,1H,J=5.1Hz), 4.38 (dd,1H), 4.20-4.13 (m,2H) 3.79-3.73 3.75 3.59-3.53 3.41 (dd,1H), 3.35-3.31 3.20 (ad,lH,J=5.1 and 14.1Hz), 2.98-2.88 1.38 -18-

CH

3

NY

R X R 535r Ana. Clc fo C28 H32 N2 011: C, 58.73; H, 5.63; N, 4.89.

Found: C, 57.37; H, 5. 77; N, 4. 78.

4 '-Dehydroxy-3 '-de:~iethoxy-3' 4'-bis-acetylamino etoposide

"ETOP'

00 6Acetic anhydride (68 iii, 0.71 mrnol) was added dropwise to a 9 99 magnetically stirred solution of diamino etoposide (product of Example 3, 0.200 g, 0.35 mmoJ.) and pyridine (60 'ple 0.74 minol) i-i CH 2 C1 2 (5 ml) at 2 0 C under an atmosphere of N 2 9 99Stirring was continued for 4 hours whereupon TLJC analysis MeOH in CH 2 C1 2 showed the presence of a new less polar product and the absence of startii~g material. The reaction mixture was poured irto water, extracteLu with three portions f OF a of CH 2 Cl 2 dried over MgS0 4 and puri.fied by flash chromatography using 4% MeOH in CH 2 C1 2 as eluent on silica gel to provide 0.111 g of off white solid, mp. (slow decomposition to a foam above 217*C).

IR (KBr) 3440 2930, 1780, 1679, 1490 cm 1 H NMR (CDC 3 6 8. 84 (s ,1H1), 7. 01 (s ,1H) 6. 80 (s,1H) 6.48 1H) 6. 47 1H) 5. 95 8Hz ,2H) 4. 94 J=3. 3TFz 4. 72 (m,1i) 4. 64 J=7. 7Hz, 1H) 4. 39 3Hz, 1H) 4. 24 J=9 9 ,2Hz ,lH) 4. 15 1H) 3. 88 3.73 (t,J=8.7Hz,lH) 3.53 (t,J=9.6Hz,1H) 3.40 (t,J=8.2Hz,1H) 3.40-3.10 (m,3H) 2.71 (bs,1E, (sugar-OH), 2.42 (bs,1H, (sugar-OH]) 2.24 (s,3H) 2.02 (s,311) .1.37 (d,J=5.lHz,3H).

-19- 11S (FAB) m/e 657 If the general procedure described in Example 4 is followed using the acylating agent listed low in place of acetic anhydride, the corresponding bis-acylated compounds are obtained.

"ET OP" C~-~r 04 00 0 '009 0~00 004 4 90, 0 0090 0 *0 o 0 0 00 Example Acylating Agent acetic formic anhydride trifluoroacetic anhydride Product R=formyl R=trifluoroacetyl a 0 0 0 0 0 0 a a0 0 00 4 0004 0 90

I

O 00 0 0 00 0 90 If the general procedure described in Example 4 is tepeated using the acylating agent listed below in a molar amount equivalent to that of 3',4'-diaminoetoposice- and in the absense of pyrid.,ne, a mixture of the corresponding 4'and inonoacylated derivatives is obtainled.

"ETO P" K-2

"ETOP"

Example Acylating Agent Product 7 acetic anhydride R=acetyl 8 acetic formic anhydride R~formy.

9 trifluoroacetic R= trif luoro anhydride ace ty 1 Example 4'-Dehydroxy-3'-demethoxy-3' ,41-bis-(4-2yridylmet'-hylene) amino Etoposide

"ETOP"

A solution of 3',41-diamino etoposide (product of Example 3t c' 181 Ing, 0.316 mmol) in dry CH 2 Cl 2 (35 ml) under N2 was treated with activated 4A molecular sieves (2.25 g) and 4-pyridine carboxaldehyde (4.56 g 42.6 mmol). The mixture was stirred at room temperature for 26 days and then applied directly to the top of a 2 cm column filled with 6 1/2 inches of neutral alumiQ-a. Sequential elution with 200 ml each of 50% and 75% EtOAc ini CH Cl 2 and then EtOAc and 10-15% CH 0129 in EtOAc removed the excess aldehyde and other :3 ixnpuritie Finally, tl'je title compound was eluted with 130 ml of CH 3 OH. After eva*.)oration in vacuo, the solids were dissolved in EtOAc (75 ml) and CH 3 OH (2 ztil) filtered, and evaporated. The r.esulting yellow solid was dissolved in 2-3% CH 3 O in CH 2 C1 2 and filtered through a 0.45 micron filter to give a clear yellow solution. 'Rotary evaporation and drying at 0.1 torr provided 145 mg, of the pure -21if title compound as a yeliovw-orange solid. The 360 MHz HNMR spectrum indicated a ca 65:35 mixture of isomers tentatively assigned as the 4'-E and the 31-E, 4'-E based purely on steric considera~tions.

Partial 1HNMR (CDCl,) 6 8. 73-8. 66 N'i,411) 8. 40 (s,1H) 8. 28 1H) 7.65-7. 57 4H) 6. 96 H) 6. 82 IH 6. 57 6.28 (d,1H) 6.01-5.94 (m,2H) 4 .77 (s,3H) 1.24 3H),1 1. 23 3H) I, S S Sr.

I, tc' If the genera,2 procedure of xexample 10 i s repeatad with the aldehydes listed below in placoe of 4-pyridinecarboxaldehyde, the correspconding bis-iino compounds are obtained.

Example

"ETOP"

Aldehyde benz aldehyde 4-methoxybenzaldehyde 3,4, 5-trimethoxybenza lde hy--Ic 3-tbioplenecarboxaldhYvie 2- furancarboxa ldehyde 2-ni trobenz aldehyde Product (R= phenyl 4 methoxypheny 1 methoxypheny 1 3-thienyl 2- fury 1 3-nitrophenyl -22- 9 Example 17 4 -Dehydroxy-3' -demfethoxy-etoposide 3' ,4'-triazole

"ETOPI"

:::Sodium nitrite (26.8 mng, 0.388 ntnol) was added to a solution of the 4'-diamino etoposide (produecr of Example 3, 169.1 mg, 0'.2953 inmol) in dry THF (4 ml) P-I glacial acetic acid (0.75 ml) The mixture was stirrP.I at room temperature for 1-rs, poured into cold saturatrA, aqueous sodium bcarbonate (100 ml), and extracted with CH Cl 2 (2x50 ml). The combined S2Rtr vprto olwe ycytliainfo extracts. were washed with brine (50 ml) and dried over CH C1 2 (3-5 ml) produced 128.1 mg of the pure tit2.e compound as a colorless solid, mp 245-250*C.

tiltIR (KBr) 3445, 1775, 1625f 1605, 1507, 1488: 1455, 1400, 1345, 1240, 1165, 1100, 1085, 1045, 1010, 945, 880, 772, 705 -1 UV (CH 3 OH) Xrnax 287 rnm (log E 3.909).

H NMR (CDCl 3 6 7.25 6.84 6.66 6.51 (s,111) 5.98 (sv',2H) 4.91 (d,C,J=3.3Hz) 4.78 (d,IH,J=5.3Hz) 4.73 (6_,1H,J=4.9Hz) 4.65 4.42 (ddM1) 4.20-4.14 4.04 3.73 (m,1H), 3.56 3.44 3.40-3.32 2.94 (m,1H), 1.38 (df..EJ-j/.9Hz).

(FAB) m/e 584 378 (M-sugar) -23- ~1W1 Anal. Calcd for C 28

H

29 N3011: C, 57.63; H, 5.01; N, 7.20.

Found: C, 57.81; H, 4.90; N, 7.11.

Example 18 4'-Dehydroxy-3'-demethoxy etopocide 3',4'-imidazole

"ETOP"

0 A solution of 3',4'-diamino etoposide (product of Example 3, 182 mg, 0.318 mmol) in CH 2 C1 2 (10 ml) was treated with 2 2 trimethyl orthoformate (500 mg, 4.7 mmol) and E-toluenesulfonic acid monohydrate (1.3 ag) and the mixture 0 00 was stirred at room temperature for 6 days. The result',ng 000 0P titl2 compound was collected by filtration as an off-white solid (28.7 mg, The remaining filtrate was treated 0 with trimethyl orthoformate (2 ml) and 2-toluenesulfonic acid monohydrate (8 mg) and stirred at room temperature for 12 day Following workup with ethyl acetate and aqueous SVsodium bicarbonate, flash chromatography of the resulting 0 crude material asing 5% and then 10% CH 3 OH in CH Cl 3 2 2 provided 53.6 mg of additional pure title compound.

IR (KBr) 3435, 1775, 1633, 1603, 1490, 1390, 1340, 1240, 1165, 1100, 1080, 1040, CL10, 940, 703 cm- 1 1 H NMR (d6-DMSO) 6 8.08 (broad s,1H), 7.02 (s,1H) 6.61 (m,1H) 6.54 6.48 6.r2 5.24 (m,2H), 4.95 4.72-4.68 4.56 4.29-4.22 4.08 (dd,1H), 3.86 3.50 (dd,1H), 3.38-3.03 2.9-2.85 1.23 (d, 4 -24- UV (CH 3 OH) Xmax 243 (sh) and 282 (log e= 3.765) nin.

MS (FAB) m/e 583 ,378 (r-sugar) Example 19 4' -Dehydroxy-3' -demethoxy etoposide 3' ,4 (2-methylimidazole) t X ~43 1 If the procedure of Example 18 is repeated using trimethy.

orthoacetate in plac-e of the orthoformate, the title compound is obtained.

Example 4 -Dehydroxy-3' -demetho,-y-etoposide 3' ,4'-(2,3-dimethylpyrazine)

"ETOP"

A solution oz 3',4'-diamino etopcside (product o Example 3, 225 mg;, 0.393 inmol) in dry CH 2 C1 2 (15 ml) was treated dropwise over 1 min with neat 2,3-butanedione (56 0.65 mmol). After 5-10 min at room temperature the reaction 0 00 0 ~4 I 4Q40 444 0 0040 @944 4 000I 4 44 4 0 4 04 404*44 0 I I I 1 0 4 '.4 4 4 I 4 4' mixture was cooled to 0 0 C and the product was collected by filtration, washed with cold CH 2 Cl 2 and dried to give 163 mg. of the analytically pure title compound as a white solid.

IR (KBr) 3450, 1776, 1620, 1575, 1508, 1490, 1387, 1342, 1236, 1203, 1165, 1117, 1095, 1080, 1040, 1007, 936, 892, 878, 700 cm 1H NI{R (d 6 -DMSO) 6 7.14 (d,1H,J=1.2Hz) 7. 05 (s ,1H) 6. 68 (d,1U,J=1.2Hz) 6.58 (s,1H) 6.04 (s,2H) 4. 97 (s,lH,J=3.4Hz) 4.80 (d,1H,J=5.6Hz) 4.71 (q,1H,J=5Hz) 4.55 (d,lH,J=7.8Hz) 4.31-4.21 (m,2H) 4.08 (dd,1H) 3.91 (s,3H), 3. 53-3. 46 2H) 3. 40-3. 05 4H) 2. 92-2. 83 (m,l1H) 2. 61 (s,3H) 2.56 (s,3H) 1.23 UV (CH 3 OH) Xmax (log E) 259 (4.661), 292 (3.778), 326 (3.681).

Anal. Calcd for 32342011: C, 61.73; H,5.50; N, 4.50, Found: C, 61.33; H, 5.24; N, 4.45.

Example _21 4' -Dehydroxy-3' -demethoxy etoposide 4'-ethy lthiop hos phamide "ET OP" Ethyl dichlorothiophosphate (49 jil, 0.37 mmol) was added to a solution of 3',4'-diamino etoposide (product of Example 3, 0.20 g, 0.35 mxnol) and pyridine (0.12 ml, 1.4 mmol) in -26- CH 2 C1 2 (4 ml) at room temperature. The reaction was refluxed for 2.5 hours and then stored at -10 0 C overnight.

Flash chromatography on silica gel using 3% MeOH in CE2 Cl2 provided 0.030 g of off-white solid (TLC Rf just above that of the starting diamine etoposide) as a mixture of diaste reomers.

IR (KBr) 3420, 2922, 1780, 1640, 1601, 1490 cm- 1HNMR (CDC 3 6 8. 61 (m,l1H) 7 (m,1 H) 7. 31 1,H) 6. 79, 6. 78 (s,l1H) 6. 50, 6. 49 (s,l1H) 5. 97 2H) 5. 86, 5. 83 (s,1H) 5,59-5. 42 2H), 4. 88 (m,l1H) 4. 73 1H) 4. 54 2H) 4. 40 (m,E 1,H 4. 17 21R) 97 (m ,2H) 73, .72 2H), 3. 44 3. 31 3 27 3H), 2. 87 1.36 (d,J=2.4Hz,3H) 1.22 I I I 1140 0001 'too to to lift 1 00 0 00 1ff 400 0 0 MS (FAB) m/e 678 M.

Example 22 4 1 -Dehydroxy-3 '-demethoxy etoposide 31 4 1 -ethylphosphamide "1ETOP 1 The procedure of Example 21 is repeated using ethyl dichlorophosphate in place of ethyl dichlorothiophosphate to provide the title compound.

-27-

Claims (10)

1. A compound having the formula Y X 00° 0 0 wherein Y is H and A is selected from the Group consisting of (C1- 10 )alkyl; (C2- 10 )alkenyl; (C 5 _6)cycloalkyl; ?-furyl; 2-thienyl; aryl; aralkyl; and aralkenyl wherein each of the aromatic rings may be unsubstituted or substituted with one or more groups selected from halo, (C_1 8 )alkyl, (C 1 8 )alkoxy, hydroxy, nitro, and amino; or A and Y are each (C 1 8 )alkyl; or A and Y and the carbon to which they are attached join to form a (C 5 6 cycloalkyl group; and B is selected from the group consisting of N 3 OR 1 HR 4 H3CO N=CHR 1 CO N NOR NHR 3 HR rk/ A 3 C04 krCtj tN~S.i C' 0 3k A 3 C 0 o 19 9* 4 @999

4.

9.9, 4.94 9 4 99 9 4I 4 I 4 9 49 9 4.4 9 9 4 9 4 4. 44 9 I oad $c0 I"wX 1 2 wherein R and R are independently selected from the group 3 consisting of (C 1 5 )alkyl, aryl, and aryl(C 1 5 )alkyl; R anc R are independently H, (C 1 5 )alkanoyl, or halo-substituted (C 2 )alkanoyl R 5 is aryl, aryl substituted with one or more groups selected from (C 1 -5)alkoxy and nitro, or heteroaryl; R 6 and R7are each H or (C 1 5 )alkyl; R 8 is (C 1 5 )alkyl or (C 1 5 )alkyyl substituted with one or more groups selected from the group consisting of hydroxy, alkoxy, alkanoyloxy, cyano, amino, alkylamino, dialkylamino, carboxy, alkylthio, niercapto, alkanoylamino, alkanoyl, carbamoyl, and halo; and X is oxygen or sulfur. 29 -j1 a P 4 48 444 44849 4844 9084 81( k4 4 4i 1 *1 C 4 NI 4 4 2. Thecompound of claim 1 wherein Y is H and A is methyl or 2-thienyl. 3. The compound of either one of claims1 or 2 wherein A is methyl. 4. The compound of claim 3 wherein B is wherein R and R 2 are as previously defined. The compound of either one of claims 3 or 4 wherein R and R 2 are both methyl. 6. The compound of either one of claims 3 or 4 wherein R 10 and R 2 are both phenylmethyl. 7. The compound of claim 3 wherein B is or a pharmaceutically acceptable acid addition salt thereof. 8. The compound of claim 3 wherein B is 4i wherein R 3 and R are the same and are selected from the group consisting of (C 1 5 )alkanoyl and halo-substituted (C2- 5 )alkanoyl. 9. The compound of either one of claims 3 or 8 wherein 3 4 R and R are each acetyl. 30 -18- i I 10. The compound of claim 3 wherein B is wherein R5 is as previously defined.

11. The compound of either one of claims 3 or 10 wherein R 5 is 4-pyridyl.

12. The compound of claim 3 wherein B is c 0

14. The compound of claim 3 wherei B is where R6 7i are independently. The compound of either one of claims 3 or 14 wherein R6 and R7 are both methyl. S* 3 13. The compound of claim 3 whereir, B is 6 7 14. The compound of caim 3o where isin R and R are both methyl. 31 4 16. The compound of claim 3 wherein B is 0 ~'INH wherein X and R 8 are as previously defined.

17. The compound of either one of claims 3 or 16 wherein R 8 is (C

18. The compound of any one of claims 3, 16 or 17 wherein X is sulfur and R 8 is ethyl. a 0 6

19. A method of inhibiting tumor growth in a mammal which comprises administering to said tumor-bearing mammal an effective amount of a compound of claim 1. 4° 20. A pharmaceutical composition comprising an effective antitumor amount of a compound of claim 1 and a pharmaceutically acceptable carrier.

21. A compound as claimed in claim 1, substantially as hereinbefore described with reference to any one of the examples. DATED: 9 December, 1988 PHILLIPS ORMONDE FITZPATRICK Attorneys for: -BRISTOL-MYERS COMPANY- I (1269C) CA IO'f See. U 17 CA